SBIR Phase I: Novel Microarray Platforms For Detection Of Rare Molecules In Complex Mixtures

这个小型企业创新研究(SBIR)第一阶段项目建议构建一个新的微阵列平台，该平台具有高蛋白质结合能力，允许增强荧光检测。现有微阵列表面的局限性包括基于平台的光学干扰和对生物分子有限或无效的结合能力。这些限制了安装在现有微阵列表面上的结合反应的最终灵敏度。我们将通过两种方式构建复合曲面或修改曲面。首先，通过将硝化纤维(一种能够基本上不可逆地结合许多不同生物分子的聚合物)浇注在光学透明的多孔径迹蚀刻膜上。我们相信这种新的表面将保持这两种起始材料的一些性质。通过改变径迹蚀刻膜的孔结构，我们将优化得到的膜捕获复杂蛋白质混合物的能力，并允许进行灵敏的荧光检测。其次，我们将直接用功能硅烷修饰径迹蚀刻膜，以提供允许蛋白质和核酸共价偶联的化学基团。这种方法应该证明有利于保持原始轨道蚀刻结构的光学兼容性。这种修饰的、光学透明的径迹蚀刻膜可能是抗体阵列的最佳选择，在抗体阵列中，捕获分子可以以足够的密度固定，以提供敏感的分析表面。该项目的更广泛的影响/商业潜力将是为一种新的分析工具提供基础，该工具将能够建立以抗体和抗原为基础的分析，从而提高灵敏度。它还将提供一个创新的表面，以定量捕获复杂混合物的生化成分，从而允许检测稀有分子。微阵列在生物科学研究、疾病和药物发现过程中以及在人类和动物诊断中发挥着越来越重要的作用。它们提供从少量宝贵的临床和研究样本中提取多个值所需的并行处理工具。具有更高灵敏度的微阵列将允许检测可能涉及细胞调节、细胞分化和疾病机制的稀有生物分子。逆相蛋白质阵列(RPPA)对于了解细胞在各种疾病状态下的变化很重要。例如，在癌症中，少量肿瘤细胞的裂解产物可以在表面被发现，然后用许多不同的抗体进行询问，以阐明这些肿瘤细胞群体中的蛋白质表达模式。通过拥有一个能够支持最灵敏的分析的平台，这些技术的能力将大大增强。

This Small Business Innovation Research (SBIR) Phase I project proposes to construct a new microarray platform with high protein binding capacity that allows for enhanced fluorescence detection. Limitations of existing microarray surfaces include platform-based optical interferences and limited or ineffective binding capacity for biomolecules. These limit the ultimate sensitivity of binding reactions mounted on existing microarray surfaces. We will construct a composite or modified surface in two ways. First, by casting nitrocellulose (a polymer able to bind many different biomolecules essentially irreversibly) on an optically transparent porous track-etched membrane. We believe this new surface will maintain some properties of both starting materials. By varying the pore structure of the track-etched membrane, we will optimize the resulting membranes ability to capture complex protein mixtures and permit sensitive fluorescent detection. Secondly, we will directly modify the track-etched membrane with functional silanes to provide chemical groups permitting covalent coupling of proteins and nucleic acids. This approach should prove beneficial to maintain the optical compatibility of the original track-etched structure. This type of modified, optically transparent track-etched membrane may be optimal for antibody arrays where the capture molecule can be immobilized at a sufficient density to provide a sensitive assay surface. The broader impact/commercial potential of this project will be to provide the basis for a new analytical tool that will allow establishment of antibody- and antigen-based assays of enhanced sensitivity. It also will provide an innovative surface to capture quantitatively the biochemical components of complex mixtures in such a way as to permit the detection of rare molecules. Microarrays play an increasingly important role in bioscience research, disease, and drug discovery processes as well as in human and animal diagnostics. They provide parallel processing tools required to extract multiple values from small amounts of precious clinical and research samples. Microarrays with enhanced sensitivity will permit the detection of rare biomolecules that may be involved in cellular regulation, cellular differentiation, and disease mechanisms. Reverse phase protein arrays (RPPA) are important for understanding cellular changes in a variety of disease states. In cancer, for example, lysates from small numbers of tumor cells can be spotted on a surface and then interrogated with many different antibodies to elucidate protein expression patterns in these tumor cell populations. The power of these techniques will be enhanced significantly by having a platform able to support the most sensitive assays.